(1) Mechanisms of virulence acquisition in Ngari virus (NRIV): In 1997-8 a large outbreak of Rift Valley fever (RVF) occurred in Kenya and Somalia. During this outbreak, NRIV was identified as the causative agent of hemorrhagic fever (HF) in a significant proportion of the cases. NRIV has been recognized as a naturally occurring genetic reassortant between Bunyamwera virus (BUNV; L and S segments) and Batai virus (BATV; M segment), both of which belong to the Bunyamwera serogroup in the genus Orthobunyavirus. Interestingly, both parental viruses cause febrile illness, but not severe HF in humans. This is a perfect example of the important role that genetic reassortment plays in the evolution of viruses and of the changes in virulence that can result. Therefore, we are using NRIV as a model to understand the molecular mechanisms underlying the emergence of novel pathogenic bunyaviruses in nature. We have recently determined the full genome sequences of 5 NRIV isolates (genotype: L-BUNV/M-BATV/S-BUNV), including 2 strains from the Kenya/Somalia HF outbreak, as well as 5 BATV isolates, including the UgMP-6830 strain, which is the closest relative of the NRIV M-segment, and 9 BUNV isolates. Based on the BUNV sequencing data obtained we have identified two distinct virus lineages, one containing viruses related to the prototype strain and another containing strains related to the NRIV progenitor. In addition, as a result of these studies we have identified a novel reassortant of BUNV, the existence of which serves to emphasize that the frequency of such viruses in nature is probably much higher than is currently appreciated. In order to gain insight into factors that facilitate reassortment in nature, we have conducted in vitro reassortment assays between BUNV and BATV, as a model for the emergence of NRIV during co-infection. We found that while nearly all possible reassortants were recovered, and thus are viable, both reassortment frequency and the products of reassortment were influence by the cell type used (mammalian vs arthropod). Combined with phenotypic data from previous experiments in which we analyzes the growth of BUNV and BATV in various relevant cell lines, we are now assessing the characteristics of these reassortants to establish a relationship between these traits and specific gene products/segments. This information will be used to guide our future studies of reassortment using the NRIV reverse genetics system, which we are in the process of establishing, as well as being used as a framework to understand the potential public health impact of other naturally occurring reassortant viruses, such as that identified as part of our broad-scale phylogenetic analysis of the Orthobunyavirus genus. (2) Genetic characterization and evolutionary modelling for orthobunyaviruses and uncharacterized bunyaviruses: The Bunyaviridae is an unusually large and diverse virus group, with the genus Orthobunyavirus alone containing more than 150 named viruses, among which are numerous human pathogens. In the absence of a sufficient body of genetic data to facilitate molecular identification, orthobunyaviruses have historically been classified into one of 18 distinct serogroups, again reflecting the high degree of genetic diversity between these viruses. However, this approach has significant limitations in that it usually focuses on only one or at most 2 viral antigens, making it much less likely that reassortants will be identified using this approach, particularly in the case of reassortants within a serogroup. In addition, serological cross-reactivity can be high among certain viruses, preventing a definitive identification based on such methods. Alternatively, some uncharacterized bunyaviruses do not show sufficient reactivity to any known group and as a result their exact nature remains unknown. However, these viruses are particularly important to our understanding of the genetic diversity that exists in nature and may also provide critical links in our understanding of the evolution of this group. In order to address these gaps in our knowledge and generate datasets of sufficient size and completeness for evolutionary analysis, we have undertaken large-scale sequencing of various orthobunyavirus groups, including the Bwamba/Pongola and Nyando group viruses, which are prevalent but understudied causes of febrile illness throughout much of Africa. Based on our sequencing efforts we have clarified the taxonomic relationships of these viruses, as well as identifying two previously uncharacterized bunyaviruses that are related to the Nyando virus group (i.e. Moju dos Campos and Kaeng Khoi viruses). These findings showed that this virus groups covers a much larger geographical area than was previously appreciated (including both Asia and South America) and may involve host species that had not previously been recognized to play a role for these virus groups. Similarly, we are now completing a study aimed at examining the distribution of Guaroa virus, a neglected cause of febrile illness in South America that seems to be undergoing expansion of its endemic area to include countries such as Peru and Bolivia. Based on our data we have defined the degree of genetic diversity of these viruses to establish broadly cross-reactive RT-PCR primer sets and generated datasets sufficient to allow phylogeographic modelling of GROV spread within South America and allowing us to identify regions of active spread that should be focused on as a part of future surveillance efforts. Additional studies focusing on the Bunyamwera group viruses are on-going. (3) Molecular characterization of tick-borne phleboviruses potentially causing human disease: In order to better understand the relationships between the molecular biological characteristics of uncharacterized viruses and their zoonotic potential, as well as their evolution, we are conducting an extensive genetic analysis and biological characterization of uncharacterized taxonomically ungrouped bunyaviruses associated with ticks, including those isolated from Africa, Asia, South and North America. During 2005-2013, Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) and Heartland virus, two novel tick-borne phleboviruses, were first recognized as the causes of severe illness with thrombocytopenia among humans in China, Japan and South Korea, or the United States, respectively. Although these tick-borne phleboviruses (TBPVs) comprise a related group in the genus Phlebovirus, along with the Bhanja group viruses (BHAVs) and Uukuniemi group viruses (UUKVs), the epidemiological study and diagnosis of all TBPVs simultaneously has been difficult due to the high degree of serological and genetic divergence among these viruses. Therefore, based on available TBPV genome sequences, determined by us and others, we have developed an RT-PCR system targeting a conserved sequence motif in the L genome segment that is able to detect the L segment RNA of all known TBPVs. This RT-PCR assay allowed us to identify some taxonomically unassigned bunya-like viruses as TBPVs. Furthermore, by collaborating with Hokkaido University and Yamaguchi University in Japan, we have discovered novel TBPVs in Japan, Mali, Zambia, and the United States using our RT-PCR system. Phylogenetic analysis revealed that viruses associated with ticks are more divergent that was previously appreciated and represent the dominant virus type within the genus Phlebovirus, as compared with mosquito/phlebotomus-borne viruses (e.g., Rift Valley fever virus).